Abstract

An impaired ability to regulate the activation of microglia by fractalkine (CX3CL1) leads to persistent neuroinflammation and behavioral alterations following lipopolysaccharide (LPS) challenge. While these responses are usually transient, LPS injection caused prolonged depressive-like behavior in fractalkine receptordeficientmice (CX3CR1(-/-)) that was associated with exaggerated microglialactivation and induction of the tryptophan (TRP) degrading enzyme indoleamine 2,3-dioxygenase (IDO). IDO activation and subsequent generation of neuroactive kynurenine metabolites may have a pivotal role in the development of depression. Therefore, the purpose of this study was to determine the extent to which LPS-induced depressive-like behavior in CX3CR1(-/-) mice was dependent on IDO activation. CX3CR1(-/-) mice were implanted prior to LPS challenge with a slow release pellet of 1-methyl-tryptophan (1-MT), a competitive inhibitor of IDO. Here we show that the depressive-like behavior evident in CX3CR1(-/-) mice 72 h after LPS injection was abrogated by inhibition of IDO. LPS also decreased body weight and locomotor activity in CX3CR1(-/-) mice, but these effects were independent of 1-MT. Consistent with the increased metabolism of TRP by IDO, the ratio of 3-hydroxykynurenine (3-HK) to TRP was increased in the brain 72 h after LPS. Increased serotonin (5-HT) turnover was also evident in the brain. The LPS-associated increases in both 3-HK:TRP and 5-HIAA:5-HT ratios were prevented by the inhibition of IDO. Last, IDO blockade attenuated microglialactivation in the prefrontal cortex and hippocampus 72 h after LPS. Collectively these data indicate that LPS-induced IDO activation contributes to persistentmicroglialactivation and depressive-like behavior in CX3CR1(-/-) mice.

LPS increased the ratio of KYN to TRP in the plasma independent of 1-MT

CX3CR1−/−mice were implanted s.c. with either placebo or 1-MT filled pellets three days prior to i.p. injection of saline or LPS (0.5 mg/kg). Plasma was collected 72 h later and concentrations of (A) 1-MT, (B) KYN, and (C) TRP were determined. (D) The ratio of KYN to TRP in the plasma is shown. Means with different letters (a or b) are significantly different (p < 0.05) from each other.

LPS increased the ratio of KYN to TRP in the brain independent of 1-MT

CX3CR1−/−mice were implanted s.c. with either placebo or 1-MT filled pellets three days prior to i.p. injection of saline or LPS (0.5 mg/kg). Whole brain was collected 72 h later and concentrations of (A) 1-MT, (B) KYN, and (C) TRP were determined. (D) The ratio of KYN to TRP in the brain is shown. Means with different letters (a or b) are significantly different (p < 0.05) from each other.

LPS-induced increase in the ratios of 5-HIAA to 5-HT and 3-HK to TRP in the brain were reduced by 1-MT

CX3CR1−/−mice were implanted s.c. with either placebo or 1-MT filled pellets three days prior to i.p. injection of saline or LPS (0.5mg/kg). Brains were collected and TRP and several other TRP-related metabolites were determined 72 h later. A) 5-HIAA:5-HT and B) 3-HK:TRP ratios are shown. Bars represent the mean ± SEM. Means with different letters (a or b) are significantly different (p < 0.05) from each other.

LPS-inducedmicroglialactivation in the prefrontal cortex (PFC) and hippocampus (HPC) of CX3CR1−/−mice was attenuated by 1-MT

CX3CR1−/−mice were implanted s.c. with either placebo or 1-MT filled pellets three days prior to i.p. injection of saline or LPS (0.5 mg/kg). Brains were collected, fixed, and processed 72 h later A) Representative images of Iba-1 staining in the PFC (top panels) and in the CA1 region of the hippocampus (bottom panels) are shown. Scale bars represent 50 μm. For each region there are 40x images that are inset (top left corner) to show selected microglia. Proportional area of Iba-1 staining in the B) PFC and C) HPC are shown. Bars represent the mean ± SEM. Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other.

Proposed Role of the IDO pathway in Depressive-like behavior following LPS challenge in CX3CR1−/−mice

In micedeficient of the fractalkine receptor (CX3CR1−/−), LPS causes amplified microglialactivation with prolonged and exaggerated production of pro-inflammatory cytokines. These inflammatory cytokines activate IDO, which in turn, shifts tryptophan metabolism away from the production of serotonin (5-HT) and towards the production of kynurenine (KYN). 5-HT can be degraded to 5-HIAA by monoamine oxidase (MAO) or by IDO to 5-hydroxykynuramine. Active KMO in microglia converts kynurenine into 3-HK, which is a pro-oxidant. This also effectively shifts KYN away from the production of KYNA. 3-HK can then be degraded into quinolinic acid (QUIN) by kynureninase. QUIN is an NMDA receptor agonist and pro-oxidant. The influence of 1-MT on this pathway is also shown. The inflammatory associated changes in TRP metabolism are highlighted using red arrows and boxes. Overall, it is hypothesized that the IDO-initiated increases in tryptophan affect both serotonergic and glutamatergic neurotransmission and leads to the development of depressive-like behavior.